Investigation of DNA Modifying Enzymes by Computational Simulations: Development and Applications Project Summary The accurate synthesis, maintenance, repair, and modification of DNA is crucial for organismal survival since errors in DNA can lead to the onset of different diseases. Therefore, enzymes related to DNA transactions need to perform their activities accurately and efficiently. Mutations arising from exogenous or endogenous factors can result in changes that affect the structure and/or function of these enzymes. There are a large number of enzyme families involved in the synthesis, repair and modification of DNA. Two of these families involve DNA polymerases (DNA pols) and AID/APOBEC enzymes. The former family includes over 16 human DNA pols, which are responsible for the accurate synthesis and repair of DNA. The AID/APOBEC enzymes comprise several members, including A3G and A3H, are involved in targeted deamination of DNA bases, and are key players in immune response. Understanding the detailed structure, function and mechanism of native and mutant versions of these enzymes can help in myriad ways, from insights on basic biochemical issues such as inter-molecular interactions to information that can aid in the development of diagnostic and/or therapeutic treatments. Computational simulations based on classical molecular dynamics (MD) and hybrid quantum mechanical (QM)/molecular mechanical (MM) methods have been shown to provide a very important tool to investigate the reaction mechanism of enzymes with atomic level detail. Our long-term goal is to develop accurate QM/MM methods to understand the mechanism, structure and function of enzymes involved in DNA modification by means of computational simulations. To this end, the goals of the present proposal are: i) To use MD and QM/MM simulations to study the structure/function/reactivity of wild type and selected mutants, including cancer variants, of two DNA Pols (DNA Pol III, and DNA Pol ?), and one APOBEC enzyme (A3H). ii) To continue the development of LICHEM, our QM/MM software, which interfaces QM programs with advanced anisotropic/polarizable force fields (GEM and AMOEBA) to accurately describe the MM environment; and to extend the QM/MM--minimum free energy path (QM/MM--MFEP) method for anisotropic/polarizable potentials to enable efficient free energy calculations for QM/MM simulations. The detailed understanding of the structure, function and reaction mechanism of the selected DNA pols and APOBEC3H will provide insights into effects of cancer mutants, as well as possible routes to develop inhibitors for these enzymes. Our collaborators, Profs. Penny Beuning, David Rueda and Rahul Kohli, will perform experimental studies based on our computational results. The successful completion of the proposed project will provide an accurate computational tool for the calculation of enzyme reactions, and the generation of structural and mechanistic insights on two important families of enzymes, that may be used to enhance the efficacy of cancer treatments.